Amino acids mutations

A classical example of sequestration is the monarch butterfly Danaus plexip-pus (Danainae) which feeds on leaves oiAsclepias curassavica (Asclepiadaceae) and sequesters cardenolides such as calotropin (158) (Fig. 29). This molecule was shown to afford the butterfly an efficient protection against birds [150-152]. It was also demonstrated that its resistance to cardiac glycosides is due to a single amino acid mutation in the ouabain-binding site of the Na+/K+ ATPase [153,154]. 

When these acidic residues where mutated to alanine (neutral) or lysine (positive), rates of inactivation were affected. The largest effect took place with the positive amino acid mutation. The authors found that mutations in the inactivation peptide are tied to those in the Tl-Sl linker near the T1 domain in other words, the inactivation peptide must be near the Tl-Sl linker when it inactivates the pore. The conclusion reached is that since the inactivation peptide cannot fit through the center of the TI4P4 complex, it must reach the ion conduction pore through lateral openings above the T1 tetramer. 

Cytochrome c folding and electron transfer are related topics. An improperly folded protein—engendered through amino acid mutation for instance— will exhibit diminished or nonexistent electron transfer. More reports will be forthcoming from many research groups on the topics of protein folding and electron transfer between proteins. The reader should consult the literature for updates on these related hot topics. The reader is referred to the 2006 Chemical Reviews article from the Bertini group (reference 101), which exhaustively surveys the cytochrome c field. 

The third relatively well-characterized genetic polymorphism is that of CYP2C9. Two well-characterized variants of this enzyme exist, each with amino acid mutations that result in altered metabolism. The CYP2C9 2 allele encodes an Argl44Cys mutation, exhibiting impaired functional interactions with P450 reductase. The other allelic variant,... 

Craig, S., Hollecker, M., Creighton, T.E., and Pain, R.H. 1985. Single amino acid mutations block a late step in the folding of fl-lactamase from Staphylococcus aureus. J. Mol. Biol. 185 681-687. 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

X. HYBRIDIZATION OF AMINO ACID MUTATION AND MODIFIED-HEME... 

As mentioned above, both the point-mutation on the distal side of the Mb and the modification of the heme-propionate side chains are effective to convert the Mb into peroxidase and peroxygenase. Thus, one can imagine that the combination of an amino acid mutation and a modified-heme reconstitution may more effectively allow us a new catalyst by oxygen storage. Recently, two examples, which demonstrated the hybrid modification of Mb, have been reported. One is the T67R/S92D Mb reconstituted with the modified hemin where a histidine is linked at the terminal of the heme-propionate side chain (108). The peroxidase activities toward p-hydroxyphenylpropionic acid and tyramine oxidations by the reconstituted mutant Mb are increased by 24- and 2.3-fold, respectively, based on the kciJKm value compared to those observed for the native Mb. 

These investigations also showed that the conversion of ECB to ECB nucleus would proceed more rapidly if ECB were first solubilized in a suitable solvent such as methanol or acetone. However, if the concentration of solvent was too high, the enzyme activity was reduced. Ideally, the enzyme itself could be tailored to suit the industrially preferred conditions (e.g., to make it more resistant to solvent or active at a different pH). One method for achieving this is to use directed evolution [42], whereby genes encoding the enzyme are mutated, screened and then recombined in vitro. Although the contributions of individual amino acid mutations are small, the accumulation of multiple mutations by directed evolution allows significant improvement in the biocatalyst for reactions on substrates or under conditions not already optimized in nature. This approach was used by Arnold and Moore [43] to make a 150-fold improvement in the activity of a -nitrobenzyl esterase in the presence of 15% DMSO. 

Thus, we concluded that Asp76 is the binding site for the basic substrate, trypsinogen, and that a single amino acid mutation (Asp76 —> Ser76) is sufficient to alter the substrate specificity of the enzyme for trypsinogen activation [29],... 

Mammen JS, Pittman GS, Li Y, Abou-Zahr F, Bejjani BA, Bell DA, Strickland PT, Sutter TR. Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (—)benzo[a]pyrene-7R- r , s-7,8-dihydrodiol. Carcinogenesis 2003 24 1247-1255. 

Interactions and the Effects of Amino Acid Mutations on Their Energetics. The Importance of Water Molecules in the Binding Epitope. 

Coveil DG, Wallqvist A (1997) Analysis of protein-protein interactions and the effects of amino acid mutations on their energetics. The importance of water molecules in the binding epitope, J Mol Biol, 269 281-297... 

Structural mutations in COL1A1 or COL1A2 produce abnormal type I collagen with substitutions, deletions, or insertions of amino acids. Mutations of this group usually cause type II, III, or IV OI (Byers and Cole, 2002). However, patients with phenotype closer to type I OI were also reported (e.g., one with the same Gly238Ser substitution as patient 1). By far the most common type of structural mutations (>80%) is substitutions of obligatory glycines as in patients 1 and 2 these mutations are the focus of this chapter. 

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